Hedgehog (HH) signaling acts throughout embryonic development to ensure proper tissue patterning and organ formation, while in adults HH signaling is vital for tissue maintenance and homeostasis. Aberrant HH pathway activity contributes to numerous developmental diseases and drives the initiation and progression of several cancers, including basal cell carcinoma, rhabdomyosarcoma, and medulloblastoma. More recently, de-regulated HH signaling has been implicated in multiple additional cancers, including pancreatic cancer, where tumor cell-derived HH ligands signal in a paracrine manner to the surrounding microenvironment. In particular, pancreatic cancer is characterized by an extensive HH-responsive fibrotic stroma that promotes tumor growth. However, strikingly little is known about the mechanisms by which HH signaling acts in pancreatic cancer. We propose to address this critical gap in our knowledge through a systematic analysis of the requirement for dosage-dependent HH signaling in pancreatic tumor growth. Through a collaborative effort, the Allen and the Pasca di Magliano laboratories have determined that altering the dosage of HH signaling through targeting of the HH co-receptors GAS1, BOC and CDON differentially regulates pancreatic tumor growth. Surprisingly, pancreatic fibroblasts lacking two of these co-receptors (GAS1 and BOC) have increased tumor-promoting ability compared to wild-type pancreatic fibroblasts when co-injected with tumor cells in mice. In contrast, pancreatic fibroblasts lacking all three co-receptors are unable to promote pancreatic tumor growth. These data suggest that the dosage of HH signaling dramatically affects pancreatic tumor growth. Further, these findings have important clinical implications, and provide an explanation for the negative outcome of recent clinical trials of HH pathway inhibitors in pancreatic cancer. Thus, the central question in this proposal is: how do different levels of HH pathway activity affect pancreatic tumor growth? This proposal continues a unique collaboration between the Allen and Pasca di Magliano labs that combines expertise in HH signaling and pancreatic cancer to test the hypothesis that the level of HH pathway activity is an essential regulator of pancreatic tumor growth. Thus, the objective of this proposal is to define the role of dosage-dependent HH signaling in pancreatic cancer through the dissection of the requirement for 1) HH ligands, 2) HH receptors and 3) downstream HH signal transduction components in pancreatic tumor growth. The expected outcomes of this work include: 1) a comprehensive analysis of the requirement for different levels of tumor-derived HH ligands in pancreatic tumor growth, 2) the elucidation of the relative contributions of cell surface HH receptors and antagonists to stromal promotion of pancreatic cancer, and 3) a determination of the effects of altered downstream HH pathway activity on tumor growth and metastasis. We expect that these results will define the role of HH signaling in pancreatic tumor growth and will provide insight into novel approaches to successfully target the HH pathway in the treatment of pancreatic cancer and other HH-driven pathologies.